The present invention relates to a method of potentiating the activity of an antibacterial agent by using an aminoglycoside, and to novel compositions comprising an antibacterial agent and an aminoglycoside. It also relates to a method of treating bacterial infection by administration of the composition of the invention. More particularly, the invention relates to the use of an aminoglycoside to potentiate the activity of antibacterial agents acting at or near cell wall sites, such as xcex2-lactams or cephalosporins. The invention also contemplates optimisation of the efficacy of aminoglycosides.
A major problem in treatment of infections caused by bacteria, particularly hospital acquired infections, is that an increasing number of bacteria are becoming resistant to antibiotics. For example, many strains of Staphylococcus and Enterococcus are now resistant to most of the currently-available antibiotics. Other organisms, such as Pseudomonas, respond poorly. This problem is exacerbated by the ability of many bacteria to transfer resistance to other species of bacteria.
In laboratory testing, this manifests itself as a requirement for concentrations of antibiotics which are higher than the reported minimum inhibitory concentration (MIC) to inhibit the growth of the organisms.
One group of antibiotics which are clinically becoming less useful due to acquired resistance are the cephalosporins. Cephalosporins are conventionally believed to act at surface sites on the bacterial cell wall at or near the enzymes responsible for cell wall synthesis. In Gram-negative organisms with an outer cell wall, the action of cephalosporins is limited by access to these surface sites in the inner cell wall because of molecular size and other determinants of ability to penetrate porin structures in the outer cell wall, and by the action of enzymes (cephalosporinases) which break down the cephalosporins. These cephalosporinases are largely responsible for the emerging clinical resistance of bacteria to cephalosporins.
Although aminoglycoside antibiotics are active against a wide spectrum of organisms, their use has been severely limited by the toxic side effects which occur at the doses required to achieve the desired antibacterial effect.
Thus, there is a need to improve the efficacy of antibiotics, particularly cephalosporins. Three is also a need to reduce the toxicity of aminoglycoside antibiotics, particularly gentamicin.
It has conventionally been thought that aminoglycosides exert their antibacterial effects via a strictly intracellular mechanism involving inhibition of ribosomal activity. However, the present inventor has examined data on uptake of radioactivelyxe2x80x94labelled aminoglycosides, and now proposes that aminoglycosides also act at the cell surface so as to contribute to the process of entry into the cell. Thus the hypothesis underlying the present invention is that an important part of the action(s) of aminoglycoside antimicrobials involve creation of breaches in external cell walls of bacteria and in other external capsular layers or membranes composed of lipopolysaccharide or mucopolysaccharide constituents.
It was thought
1. that the exposure profiles necessary for this action of aminoglycosides were likely to differ from the concentration-time profiles found to apply to intracellular effect(s), and that novel exposure profiles might be identified which would allow avoidance of toxicity on mammalian systems;
2. that the breaches in external cell walls and capsular membranes and layers of bacteria could facilitate entry and access to sites of action of other antibiotics such as cephalosporins which acted at or near cell surfaces, and additionally, that enzyme degradation of antibiotics (e.g. cephalosporinases) might be by-passed.
It has now been surprisingly found that the activity of xcex2-lactam antibiotics, including cephalosporins, can be potentiated by the use of a non-toxic amount of an aminoglycoside antibiotic.
The studies detailed herein, using gentamicin and tobramycin demonstrate that the concentration-time profiles producing the cell surface effect involve relatively prolonged exposures over many hours, at lower concentrations than those normally used clinically, where rapid onset (bolus) of high concentration exposure has been the characteristic approach to clinical dosing.
The potentiation of cephalosporin action by degrees in excess of 100 fold was also a surprising finding and suggests the efficiency of cell wall porin as exclusion barriers and the enzymatic (cephalosporinase) destruction of cephalosporins.
BRIEF DESCRIPTION OF THE INVENTION
In one aspect, the invention provides a method of potentiating the activity of an antibacterial agent active on bacterial cell wall, comprising the step of administering to a subject in need of such treatment said antibacterial agent and an aminoglycoside in an amount effective to attain a peak concentration of at least 4 mg/l of the aminoglycoside, and thereafter maintaining the aminoglycoside at a concentration of up to 4 mg/l for at least 1 hour.
Preferably, the antibacterial agent is active on bacterial cell wall and acts at or near the cell wall of the bacteria. Thus, the invention provides a novel action of an aminoglycoside at cell surfaces which results in the potentiation of the effect of one or more antibiotics acting at or near bacterial cell wall or sites thereof.
Preferably, the antibacterial agent is a xcex2-lactam and most preferably, a cephalosporin or cephamycin.
The aminoglycoside is most preferably gentamicin or tobramycin.
In a preferred embodiment, the activity of cephalosporin is potentiated by administering to a subject an amount of gentamicin or tobramycin effective to produce a peak concentration of up to 18 mg/l plasma. This may be achieved by administration of 70-280 mg (1-4 mg/kg body weight) over 1-2 hours. Thereafter, the aminoglycoside is administered preferably at 5-20 mg/hr for 4-12 hours to maintain a plasma concentration of 1-4 mg/l. The aminoglycoside may further be maintained at a concentration of 1.0 mg/l plasma or less up to 24 hours.
Desirably, 300 mg of cephazolin as a specific example is administered over 24 hours, maintaining a plasma concentration at 2 mg/ml or more.
In a particularly preferred embodiment, the cell wall active antibacterial agent is at 2 mg/ml when the aminoglycoside is maintained at 1-4 mg/l, and for a further 8-24 hours thereafter.
The method of the invention thus provides a critical profile of aminoglycoside exposure in the animal or human body which is necessary for optimisation of the action of aminoglycoside(s), but more particularly, for the potentiation of one or more other antibiotics. This also allows the avoidance and/or minimisation of the known clinical toxic effects of aminoglycosides on hearing, posture and dynamic balance. The profile is achieved by taking account of transfer of antibiotics from blood to tissues. However, provision is made in the method such that the antibiotic concentrations in the inner ear (vestibular apparatus and organ of Corti) do not achieve concentrations known to be toxic, and to prevent those toxic concentrations from being present for periods of time known to be necessary for toxic effects to develop. (McLean A J, Ioannides-Demos L L, Spicer W J, Christophidis N, xe2x80x9cAminoglycoside dosing: one, two or three times a day?xe2x80x9d, Med J Aust. 164:39-42, 1996).
The peak concentrations of aminoglycoside which are most desirable are 4-18 mg/l, and are preferably maintained at a minimum of 1-4 mg/l, depending on bacterial sensitivity as evidenced by MIC testing. Maintainance of this level would require a mass of some 5-15 mg per hour of gentamicin to be delivered to the circulation of an average patient at a uniform rate, although variations will be required as a result of differing body size, renal function and various disease conditions (McLean et al 1996, supra). The rate and amount of active agent to be delivered can be determined easily by a person skilled in the art.
In accordance with existing formulation protocols, such exposure would presuppose intramuscular administration of a mixture of depot formulation. However, recent developments would allow for intravenous or oral formulations. Such formulations would need to deliver initial profiles as described above (4-18 mg/l), then allow for maintainance of concentrations at 1-4 mg/l for specific periods of time. Following this, lower levels of aminoglycoside and cephalosporin may be maintained for about 8 hours onwards. In one specific embodiment of the formulation, ongoing concentrations of aminoglycoside in the circulation should not exceed about 1 mg/l at about 8-16 hours after administration of the formulation, so as to prevent toxic levels of aminoglycoside accumulating in the inner ear and kidney.
The method of the invention also allows the effective dose of the antibacterial agent which is potentiated to be reduced. This again allows toxic effects to be negated or avoided.
The method of the invention allows the development of a pharmaceutical formulation of cephalosporins such as cephazolin which are clinically effective at ⅙ to ⅓ of the current clinical doses. Physical tolerance would be enhanced markedly so that intramuscular formulations can be realistically used and tolerated. However, advances in formulation should allow the development of intravenous or oral formulations to deliver greatly reduced concentrations of the drug required as a result of the aminoglycoside potentiation.
Thus, in a second aspect, the invention provides an antibacterial composition comprising an aminoglycoside and an antibacterial agent active on bacterial cell wall, said composition formulated thereby to attain a peak concentration of at least 4 mg/l aminoglycoside which is thereafter maintained at a concentration of up to 4 mg/l for at least 1 hour following administration to a subject in need of such treatment so as to potentiate the activity of said antibacterial agent.
In a third aspect, the invention provides a method of treating bacterial infection, comprising the step of administering to a subject an antibacterial agent active on bacterial cell wall together with an aminoglycoside to attain a peak concentration of at least 4 mg/l of aminoglycoside and thereafter maintaining the aminoglycoside at a concentration of up to 4 mg/l for at least 1 hour; wherein said aminoglycoside potentiates the activity of said antibacterial agent.
The compositions of the invention may comprise a cephalosporin and an aminoglycoside in dosage-unit form and optionally, in admixture with a conventional, pharmaceutically acceptable carrier suitable for administration to a clinical or home patient by intramuscular, subcutaneous, intravenous, oral or rectal administration. The relative proportions of aminoglycoside and other antibiotics to be delivered can be determined without undue experimentation by a person skilled in the art and in view of the teachings herein.
Preferably, the compositions of the invention would allow once-a-day administration of antibiotics either in hospital or at home.
The method and composition of the invention may be used in the treatment of infection by Gram-negative, Gram-positive bacteria or mycobacteria. Other conditions include but are not limited to surgical chemoprophylaxis, and focal or systemic sepsis.
The aminoglycosides other than gentamicin which share its mechanisms of action and are comprehended by this invention include: tobramycin, netilimicin, amikacin and streptomycin.
The agents directly related to cephazolin and sharing the potentiating mechanisms directly are cephalosporins and cephamycins, as exemplified by but not limited to the following: cephalosporin, cephalothin, cephaloridine, cephalexin, cephaglycin, cephradine, cefaclor, cefoxitin, cefamandole, cefotaxime, ceftriaxone, ceftazidime and cefotetan.
The cephalosporin dosage required for this invention is far lower than has been used before, specifically 0.166-0.33 g versus 1 g current minimum standard dose (Cahn M M et al, xe2x80x9cComparative serum levels and urinary recovery of cephazolin, cephalosporin and cephalothin in humanxe2x80x9d, J. Clin. Pharmacol. 14:61-66, 1974).
Agents known generically as xcex2-lactam antibiotics share mechanisms with cephazolin, and constitute the various penicillin groups and monobactams. Examples include the following: penicillin G, ampicillin, methicillin, flucloxacillin, carbenicillin, ticarcillin, piperacillin, imipenin.
Other agents which will benefit because of improved access to sites of action include diverse agents exemplified by: bacitracin, chloramphenicol, macrolides such as erythromycin, clarithromycin, rifampicin, vancomycin, quinolonem antibiotics such as nalidixic acid, norfloxacin, cycloserine and metronidazole.
The organisms amenable to therapy by way of the various combinations include a wide variety of Gram-positive and Gram-negative organisms with a variety of growth circumstances and requirements ranging from aerobic to anaerobic growth, including:
(a) Gram-positive bacteria such as Strep.pyogenes (Group A), Strep.pneumoniae, Strep.GpB, Strep.viridans, Strep.GpD -(Enterococcus), Strep.GpC and GpG, Staph.aureus, Staph.epidermidis, Listeria monocytogenes, Anaerobic cocci, Clostridium spp., and Actinomyces spp; and
(b) Gram-negative bacteria such as Escherichia coli, Enterobacter aerogenes, Kiebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia stuartii, Serratia marcescens, Citrobacter freundii, Salmonella typhi, Salmonella paratyphi, Salmonella typhi murium, Shigella spp., Yersinia enterocolitica, Acinetobacter calcoaceticus, Flavobacterium spp., Haemophilus influenzae, Pseudomonas aueroginosa, Campylobacter jejuni, Vibrio parahaemolyticus, Brucella spp., Neisseria meningitidis, Neisseria gonorrhoea, Bacteroides fragilis, and Fusobacterium spp. and
as well as other organisms such as Mycobacterium tuberculosis, Mycobaterium smegmatis and other Mycobacteria.
Selected antibiotic combinations may be used in accordance with the invention in the following clinical conditions:
(a) Surgical chemoprophylaxis such as: ear, nose and throat surgery (otolaryngology); genitourinary surgery; contaminated penetrating injuries of the skin; compound fractures; bite wounds; penetrating eye injuries; abdominal surgery; acute cholecystitis; perforated viscus; peritonitis with cirrhosis; and dental chemoprophylaxis; and
(b) Focal and systemic sepsis such as: bacterial endocarditis; empirical therapy of systemic sepsis; skin cellulitis; decubitis, ischaemic and diabetic ulcers; severe or hospital-acquired, or institutional pneumonia; urinary infection; febrile neutropaenia; prostatitis; epididymo-orchitis; suppurative wound infections; gangrene; osteomyelitis; and pulmonary tuberculosis (for streptomycin combinations according to the infection).
In a further aspect, the invention involves the administration in specified ways of an aminoglycoside(s) to optimise the action of the aminoglycoside alone and to potentiate the action of one or more antibiotic agents which act at or near cell wall sites of bacterial cells.
When cephalosporins are placed in culture medium containing susceptible bacteria, cephalosporin diffuses down the concentration gradient existing between the concentration in the culture medium and the concentration at the sites of action on the bacterial cell wall. The action of cephalosporinases chemically degrades and inactivates the cephalosporin such that there are limitations on the degree of inhibition of growth of the bacteria.
In comparative experiments described in the examples herein, a reference strain of Escherichi coli (E. coli NCTC 10418) was exposed to constant concentrations of cephazolin in the culture medium (see FIG. 1A and 2A). As these concentrations were varied, the growth of the bacterial culture was changed. At the lowest concentration of cephazolin (1.0 mg/l) the growth of the culture was delayed in relation to control (see FIG. 1B). As concentrations in the medium were increased, there was a major decline in numbers and a delay in regrowth.
However, it was not possible to produce a complete kill or eradication of E.coli NCTC 10418 using cephalosporin alone. The organisms in the media were able to maintain their numbers in a reduced state even in the continuing presence of cephazolin. This outcome is due to the exclusion of cephalosporins by porin channels in the outer cell wall and to the action of cephalosporinase which keeps cephazolin levels below that required for full bactericidal effect.
The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the figures. The complete data shown graphically in FIGS. 1, 2 and 3 are contained in Tables 1, 2 and 3.
While a representative microorganism, E. coli, a representative aminoglycoside antibiotic (gentamicin) and a representative cephalosporin (cephazolin) were studied herein to generate the majority of data presented, it will be clearly understood that the invention is not limited to this microorganism or to these specific agents.